Animal Studies

Emerging evidence has indicated that colony-stimulating factor-1 (CSF1) modulates neuroinflammation in the central nervous system (CNS) and the development of neuropathic pain, while the underlying mechanism remains unknown. Here, we identified the increased expression of CSF1 derived from activated astrocytes in the ipsilateral dorsal horn in rats with spinal nerve ligation (SNL). Suppression of CSF1 expression alleviated neuroinflammation, neuronal hyperexcitability and glutamatergic receptor subunit upregulation in the dorsal horn and improved SNL-induced pain behavior. We also found reduced miR-214-3p expression in the ipsilateral dorsal horn following an SNL procedure; miR-214-3p directly bound to the 3'-UTR of CSF1 mRNA and negatively regulated CSF1 expression. Intrathecal delivery of miR-214-3p mimic reversed the enhanced expression of CSF1 and astrocyte overactivity and alleviated the IL-6 upregulation and pain behavior induced by SNL. Moreover, suppression of spinal miR-214-3p increased astrocyte reactivity, promoted CSF1 and IL-6 production, and induced pain hypersensitivity in naïve animals. Furthermore, SNL induced the expression of DNA methyltransferase 3a (DNMT3a) that was associated with the hypermethylation of the miR-214-3p promoter, leading to reduced miR-214-3p expression in the model rodents. Treatment with the DNMT inhibitor zebularine significantly reduced cytosine methylation in the miR-214-3p promoter; this reduced methylation consequently increased the expression of miR-214-3p and decreased the content of CSF1 in the ipsilateral dorsal horn, and further, attenuated IL-6 production and pain behavior in rats with SNL. Together, our data indicate that the DNMT3a-mediated epigenetic suppression of miR-214-3p enhanced CSF1 production in astrocytes, which subsequently induced neuroinflammation and pain behavior in SNL model rats.

Altered expression of circular RNA (circRNA) is recognized as a contributor to malignant pain where microRNA (miRNA) exerts an essential effect. We generated a murine model for bone malignancy pain in which 2472 osteolytic sarcoma cells were injected into the femurs of mice. CircRNA microarray and quantitative PCR (qPCR) and revealed that circ9119 expression was repressed in the spinal cord of bone malignancy pain model mice, which is the first relay site involved in the transmission of nociceptive information to the cerebrum of mice that receive spinal analgesics for malignancy pain. Overexpression of circ9119 by plasmid injection in the model mice reduced progressive thermal hyperalgesia and mechanical hyperalgesia. Bioinformatics prediction and dual-luciferase reporter assay showed that circ9119 functions as a sponge of miR-26a, which targets the TLR3 3'-untranslated region. Furthermore, expression of miR-26a was elevated and TLR3 level was repressed in bone malignancy pain model mice, which were counteracted by circ9119 in the spinal cord of tumor-bearing mice. Moreover, excessive expression of miR-26a was involved in the recovery of mice from progressive thermal hyperalgesia and mechanical hyperalgesia triggered via circ9119. TLR3 knockdown in bone malignancy pain model mice thoroughly impaired pain in the initial stages and reduced the effects of circ9119 on hyperalgesia. Our research findings indicate that targeting the circ9119-miR-26a-TLR3 axis may be a promising analgesic strategy to manage malignancy pain.

The cornea is highly enriched in sensory neurons expressing the thermal TRP channels TRPV1, TRPA1, and TRPM8, and is an accessible tissue for study and experimental manipulation. The aim of this work was to provide a concise characterization of the expression patterns of various TRP channels and vesicular proteins in the mammalian cornea.

In the central nervous system, the inhibitory GABAB receptor is the archetype of heterodimeric G protein-coupled receptors (GPCRs). Receptor interaction with partner proteins has emerged as a novel mechanism to alter GPCR signaling in pathophysiological conditions. We propose here that GABAB activity is inhibited through the specific binding of fibulin-2, an extracellular matrix protein, to the B1a subunit in a rat model of neuropathic pain. We demonstrate that fibulin-2 hampers GABAB activation, presumably through decreasing agonist-induced conformational changes. Fibulin-2 regulates the GABAB-mediated presynaptic inhibition of neurotransmitter release and weakens the GABAB-mediated inhibitory effect in neuronal cell culture. In the dorsal spinal cord of neuropathic rats, fibulin-2 is overexpressed and colocalized with B1a. Fibulin-2 may thus interact with presynaptic GABAB receptors, including those on nociceptive afferents. By applying anti-fibulin-2 siRNA , we enhanced the antinociceptive effect of intrathecal baclofen in neuropathic rats, thus demonstrating that fibulin-2 limits the action of GABAB agonists . Taken together, our data provide an example of an endogenous regulation of GABAB receptor by extracellular matrix proteins and demonstrate its functional impact on pathophysiological processes of pain sensitization.

PY receptors (PYRs) are among the various receptors that play an important role in nociception. The goal of this research was to investigate possible PYR expression changes in the trigeminal ganglion (TRG) in bilateral masseter muscle (MM) hypersensitivity following unilateral MM inflammation. The impact of unilateral intramasseteric administration of PYR antagonist on bilateral MM hypersensitivity was also explored.

Patients with chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) commonly experience learning and memory decline and the underlying pathogenesis remains unclear. Therefore, we aimed to study the effects of CP/CPPS on cognitive function by using a mouse model of experimental autoimmune prostatitis (EAP). Non-obese diabetic mice were immunized subcutaneously by prostate antigen and adjuvant twice and tested for cognitive performance by Morris water maze and novel object recognition test after the EAP induction. Then, dendritic complexity and spine densities were measured by using the Golgi-Cox procedure. Transmission electron microscopy was used to observe the synaptic morphology. In addition, activation of microglia and its association with synapses were also investigated by immunofluorescence staining. Our results showed that EAP induced a notable decrease in the learning and memory ability of mice, simultaneously causing a reduction in dendritic complexity detected by Sholl analysis. Likewise, the spine densities and synaptic proteins including synaptophysin and postsynaptic density protein 95 (PSD95) were significantly decreased in the EAP group. These observations were also accompanied by structural changes in synaptic plasticity. Additionally, EAP mice showed microglial activation in the hippocampus, and these activated microglia further increased contact with synaptic terminals. Taken together, our data are the first to indicate that EAP induces cognitive declines and structural neuroplastic changes in mice, accompanied by microglial activation and microglia-synapse contacts.

Although burst and high-frequency (HF) spinal cord stimulation (SCS) relieve neuropathic pain, their effects on neuronal hyperexcitability have not been compared. Specifically, it is unknown how the recharge components of burst SCS-either actively balanced or allowed to passively return-and/or different frequencies of HF SCS compare in altering neuronal activity. Neuronal firing rates were measured in the spinal dorsal horn on day 7 after painful cervical nerve root compression in the rat. Motor thresholds (MTs) and evoked neuronal recordings were collected during noxious stimuli before (baseline) and after delivery of SCS using different SCS modes: 10 kHz HF, 1.2 kHz HF, burst with active recharge, or burst with passive recharge. Spontaneous firing rates were also evaluated at baseline and after SCS. The average MT for 10 kHz SCS was significantly higher (p < 0.033) than any other mode. Burst with passive recharge was the only SCS mode to significantly reduce evoked (p = 0.019) and spontaneous (p = 0.0076) firing rates after noxious pinch. This study demonstrates that HF and burst SCS have different MTs and effects on both evoked and spontaneous firing rates, indicating they have different mechanisms of providing pain relief. Since burst with passive recharge was the only waveform to reduce firing, that waveform may be important in the neurophysiological response to stimulation.